This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ability to sort and individually interrogate specific fractions of microorganisms in the environment is hampered by the high diversity of microbes (bacteria, fungi, algae, and potentially millions of species), by our inability to culture the vast majority of microorganisms that can be observed microscopically, and by the lack of hybridization methods that are sensitive enough to detect cells that are not metabolically active or that are present in complex mixtures containing species that are autofluorescent. Recent advances in flow cytometry and cell sorting at LANL (improved sorting capability, phase instruments and acoustic focusing) could overcome some of the current limitations in use of flow sorting for collection and evaluation of microbial components of complex environments such as soils and microbial mats. Current research applications in biothreat detection, climate change, and genomics could benefit from the ability to specifically sort individual species or functional groups of bacteria out of complex mixtures. Examples include isolation of uncultured Francisella cells from aerosols, partitioning of photosynthetic and non-photosynthetic members of soil or mat communities, and fractionation of active/inactive components of complex microbial communities prior to metagenomic analysis. We propose to collaborate with the Flow Resource on Project 1, to apply the acoustic focusing and sorting technology to answer fundamental questions in environmental microbial ecology, and we present three applications for this technology, presented in order of increasing sophistication and difficulty. We will use these three projects to help drive the development of the acoustic focusing technology and its integration with existing flow techniques. Soil microbial communities are comprised of a wide variety of prokaryotic (bacteria) and eukaryotic (fungi, algae, microarthropods) assemblages. For many applications in soil microbiology it is desirable to isolate one group from the other. No current technology can successfully do this due to the wide variety of shapes and sizes represented by both cell types. Because it separates in real time, based on both size and shape, and has a very broad dynamic range, the acoustic focusing technology has potential to separate complex microbial mixtures into size classes, followed by nucleated/anucleated domains, that can help us enrich for particular components of the community. Immediate applications for this capability include reducing complexity prior to metagenome analysis, and increasing sensitivity for particular target genes or functional groups known to be represented in one of the domains. A second potential application, is to sort complex microbial mixtures from air samples. Our current evaluations of thousands of air filters in U.S. cities (through programs with BioWatch and the EPA), have documented a diverse array of bacterial species present in respirable aerosols. In some cases, we are trying to isolate particular target bacterial pathogens that are not-yet-culturable, from an abundance of spore-forming species. The acoustic focusing technology would allow us to separate the target cells from the 'contaminating'spores. In previous attempts to use flow cytometry and sorting to enrich for specific unculturable bacteria from complex environmental mixtures, we have been hampered by the tendency of different bacterial species to form aggregates, making separation of fluorescently stained vs. unstained cells difficult using tradition flow cytometry and sorting. The ability of the acoustic focusing to sort cell populations by shape as well as size, and the incorporation of lanthanide dyes that are detectable at wavelengths outside the natural autofluorescence spectrum, make it a potentially powerful tool to enrich for specifically stained microbial components. In many cases, such direct enrichment is the most promising approach for downstream genomic analysis.